Microfluidic device with controlled substrate conductivity

ABSTRACT

A method to achieve controlled conductivity in microfluidic devices, and a device formed thereby. The method comprises forming a microchannel or a well in an insulating material, and ion implanting at least one region of the insulating material at or adjacent the microchannel or well to increase conductivity of the region.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 10/384,349, filedMar. 7, 2003, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/362,340, filed Mar. 8, 2002, which is incorporated herein byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to microfluidic devices, andspecifically to methods for modifying the conductivity of materials usedin the fabrication of those devices.

2. Related Art

Microfluidic technology enables the miniaturization and automation ofmany laboratory processes. Devices employing microfluidic technology canintegrate the power of an entire laboratory full of equipment and peopleinto a single “lab-on-a-chip.” Each microfluidic device (hereafter alsoreferred to as a “chip”) contains a network of microscopic channels, ormicrochannels, through which fluids can be moved and in whichexperiments can be performed. The design of microfluidic devices forbiochemical applications involves the disciplines of fluid dynamics,biochemistry, software, and thin film manufacturing.

In microfluidic devices, the driving forces that move fluids within thechannels of the device can be electrokinetic forces, pressure forces, ora combination of the two. Electrokinetic forces are typically generatedby applying an electric field across a microchannel, where the directionof the field is parallel to the desired direction of fluid flow. Theelectric field is typically applied by placing electrodes in reservoirsat the ends of the microchannel, and applying a voltage across theelectrodes with a computer-controlled power supply. The voltage appliedacross the electrodes produces fluid flow via one or both of thephenomena of electroosmosis or electrophoresis. Electroosmosis occurswhen an electric field is applied across a channel whose surface orwalls contain charged functional groups. The charge on the channel wallionizes a thin layer of fluid near the wall. This thin layer of ionizedfluid is attracted to one of the electrodes, creating a flow of ionizedspecies toward that electrode. The flow of ionized species produces botha bulk fluid flow and an electrical current. The bulk flow rate througha microchannel can be controlled with a high degree of precision bycontrolling the electrical current that accompanies the flow through themicrochannel. The other phenomena that produces electrokinetic flow,electrophoresis, is the movement of charged molecules or particles in afluid subjected to an electric field. Electrophoresis can be used tomove charged molecules in solution, or to separate charged moleculesthat have different electrophoretic mobilities (which is roughly theircharge to mass ratio). Electrophoresis and electroosmosis often occur atthe same time when an electric field is applied to a microchannel.Techniques have been developed for minimizing one electrokinetic forcewhile maintaining the other, as appropriate, for a given application.Precise control over fluid flow within microchannels requires precisecontrol of the driving forces, such as electrokinetic or pressureforces. Precise control over fluid flow also requires preciseengineering of the microchannels themselves because fluid flow alsodepends on channel geometry and surface properties.

Microfluidic devices are typically fabricated by etching or embossinggrooves into a substrate, and then affixing a cover to the substrate toform the microchannels. In most microfluidic devices that employelectrokinetic flow, both the substrate and the cover plate are made ofan insulating material such as glass. Insulating materials help reducethe electrical current leakage between microchannels. By reducingcurrent leakage between microchannels, the use of insulating materialsallows an increased packing density of components, such asmicrochannels, in a microfluidic device.

In some applications, it may be advantageous to allow a localizedleakage of current between different channels in a microfluidic device.The leakage of current between channels allows the electrical currentsthat drive electrokinetic flow to flow in directions other than parallelto the length of the microchannels. In other words, having a conductivepath between channels provides the ability of initiating electrokineticflow in directions other than along the length of a channel. Forexample, fluid could be made to flow into the sidewall of a channel.Microfluidic devices with a conductive path between channels couldprovide advantages over standard microfluidic devices in the areas ofsample concentration and two-dimensional separation.

One set of researchers has fabricated microfluidic devices that employelectrical current leakage between microchannels for the purpose ofconcentrating samples. Khandurina, J., et al., Anal. Chem. 71, pp.1815-1819 (1999). In these microfluidic devices, the current leaksbetween microchannels through a porous membrane. The porous membrane isa separate layer of material sandwiched between the cover plate andsubstrate of a microfluidic device. In the devices shown in Khandurina,fluid from a main channel that terminates at a “T” shaped intersectionwith a separation channel. The fluid from the main channel is made toflow straight into the opposing wall of the “T” shaped intersection byallowing electrical current to flow into the opposing wall through aporous membrane above the wall. By flowing sample from the main channelinto the opposing wall, the sample accumulates, and thus concentrates,at the “T” intersection. When enough sample has accumulated at theintersection, the sample is directed to flow down the separationchannel. The device in Khandurina could be useful in assays in which asample to be separated into components must be concentrated in order toincrease the concentration of at least some of the components above adetectable threshold.

There are several problems with microfluidic devices that employ porousmembranes to provide conductive paths between microchannels. First, thelifetime of these devices is short and unpredictable due to the natureof the porous membrane. Second, the resistance of the porous membranesmay change with time. Third, the process for fabricating porousmembranes lacks the dimensional control needed to fabricate porousmembranes between closely spaced microchannels. Fourth, the nature ofthe conductivity of the porous membrane is not certain, and that couldlead to unexpected fluctuations of conductivity both between and withinmicrofluidic devices. Finally, having a conductive path betweenmicrochannels may prevent the manufacture of devices with densely packedmicrochannels.

Given the limitations of porous membranes, it is desirable to have analternative method of providing conductive paths between microchannelsin a microfluidic device. It would be particularly desirable if theconductive paths could be provided in a way that does not require theaddition of an extra layer of material, such as the above-describedlayer of a porous membrane material, to the microfluidic devicestructure. Furthermore, it would be desirable that the dimensions of theconductive paths be able to be precisely and accurately defined. Itwould also be desirable that the degree of conductivity between channelsbe controllable. In its various aspects, embodiments of the presentinvention provide these and other advantages over currently knownmethods of allowing current to flow between the channels of amicrofluidic device.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a microfluidic device comprising atleast two microchannels formed from grooves in an insulating substrate,and at least one ion implanted region in the insulating substratelocated between the grooves forming the microchannels, the at least oneion implanted region having increased conductivity compared to theinsulating substrate.

The present invention is also directed to a method to achieve controlledsubstrate conductivity in microfluidic devices, and devices formedthereby. The method comprises forming a microchannel in an insulatingsubstrate, and ion implanting at least one region of the insulatingsubstrate at or adjacent the microchannel to increase conductivity ofthe region. In some embodiments of the invention, the insulatingsubstrate is a silica-based material, whereby the ion-implanting stepincreases the conductivity of the silica-based material in at least oneregion. In alternative embodiments, the insulating substrate is apolymer material, whereby the ion-implanting step increases theconductivity of the polymer material in at least one region.

By providing regions where substrate conductivity is increased, it ispossible to run loading currents through the substrate, and thusaccumulate sample components. The ion-implantation process used inembodiments of the invention can be accurately and precisely modify theconductivity of small areas of an insulating substrate, so that theinvention is compatible may be employed on a microfluidic device withclosely packed microchannels.

These and other advantages and features will become readily apparent inview of the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings in which like reference numbers indicateidentical or functionally similar elements. Additionally, the left-mostdigit of a reference number identifies the drawing in which thereference number first appears.

FIG. 1 is an exploded view of a microfluidic device in accordance withthe invention.

FIGS. 2 and 3 illustrate methods of forming regions of increasedconductivity in microfluidic chips in accordance with the presentinvention.

FIG. 4 illustrates a microfluidic device in accordance with the presentinvention.

FIG. 5 is a magnified view of a portion of the microfluidic device ofFIG. 4.

DETAILED DESCRIPTION

Embodiments of the present invention will now be discussed in detail.While specific features, configurations and arrangements are discussed,it should be understood that this is done for illustration purposesonly. A person skilled in the relevant art will recognize that othersteps, configurations and arrangements may be used without departingfrom the spirit and scope of the invention. It should be appreciatedthat the microfluidic devices in accordance with the present inventioncan be used to perform a variety of experiments and operations, and thusthe techniques described herein could be used in connection with devicesfor such other functions.

An exploded view of a microfluidic device in accordance with theinvention is shown in FIG. 1. The microfluidic device 10 comprises twolayers: a substrate 12 and a cover plate 18. The substrate 12 may bemade of a variety of materials, including quartz, glass, polymer,ceramic or even semiconductor materials. The substrate 12 comprises apattern of grooves 16 on the upper surface 14 of the substrate. Thepattern of grooves 16, when enclosed by the cover plate 18, will formthe pattern of microfluidic channels in the assembled microfluidicdevice. The pattern of grooves may be formed by a variety ofmanufacturing methods, many of which are used in the semiconductorindustry. For example, the pattern of grooves may be formed by embossingthe pattern onto a polymer substrate, injection molding a polymer into asubstrate containing the pattern, or by a combination of lithography andetching. In one embodiment, the pattern of grooves 16 is defined using alithography process, and etched into the substrate using a wet etchprocess. A process combining lithography and wet etch is able to createhighly precise microchannels with dimensions that can be varied by widthand depth. A typical microchannel is roughly 50 μm wide and 10 μm deep.

After the pattern of grooves 16 is formed on the top surface 14 of thesubstrate 12, a cover plate 18 is fused with the substrate 12. Suchfusion can be performed using a variety of known bonding techniques,including thermal and anodic bonding. The cover plate 18 may be formedfrom a variety of materials, including quartz, glass, polymer, ceramicor semiconductor materials. The cover plate 18 encloses the pattern ofgrooves 16 formed in the substrate 12 and converts them to microfluidicchannels, or microchannels. Either or both of the substrate 12 or coverplate 18 may include holes or apertures disposed therein. In theembodiment shown in FIG. 1, holes 24 in the cover plate 18 formreservoirs or wells that are disposed above and fluidly connected to theunintersected termini of the grooves in the substrate 12. Fluids may beintroduced into the microchannels of the assembled microfluidic devicethrough these reservoirs. The size of an assembled microfluidic devicecan vary from less than one inch to a few inches on a side. Theassembled devices are typically packaged in plastic holders, which makethem easier for the user to handle.

Other embodiments of microfluidic devices are compatible with thepresent invention. For example, instead of only having holes in thecover layer, microfluidic devices in accordance with the invention mayhave holes disposed through the substrate or through both the substrateand cover plate. The extra holes could either provide separatereservoirs on opposing sides of the microfluidic device or to providethrough-holes that provide fluid access to the channels of the device.Embodiments of microfluidic devices employing holes in the substrate orboth the substrate and cover layer are in U.S. Pat. Nos. 5,779,868 and6,090,251, both of which are assigned to the assignee of the presentinvention. Other embodiments of microfluidic devices that are compatiblewith the present invention include multilayer microfluidic devices,which comprise two or more substrate layers. The microchannel formed inthe various layers of a multiplayer microfluidic device can beinterconnected using vias or through-holes.

Microfluidic devices, or chips, are currently commercially available intwo basic formats: planar and sipper. In planar chips, such as the chipshown in FIG. 1, the user introduces all chemical reagents, includingsamples, into reservoirs on the chip. Planar chips are sold for use withthe Agilent 2100 Bioanalyzer system. These chips include the LabChip®DNA Analysis, RNA Analysis, Protein Analysis and Cell FluorescenceAnalysis chips, which are manufactured by Caliper Technologies Corp., ofMountain View, Calif. Because samples are deposited into separatereservoirs in planar chips, the number of samples that can be analyzedby a planar chip is typically limited by the number of sample reservoirsprovided on that chip. While planar chips could be washed andpotentially reused, they are generally discarded after use.

Sipper chips are designed to analyze a large number of samples, whichmakes sipper chips useful for high throughput applications. In thesipper chips, minute quantities of a large number of samples can betested in a single chip. The samples are introduced into the capillaryone after the other, spaced by buffer solution. The samples then proceedthrough the microchannel network in a continuous flow, assembly-linefashion. A typical sipper chip employs one or more integrated sampleaccession capillaries for interfacing with an external collection ofsample sources, such as a multiwell plate. The sample accessioncapillary is typically a small glass tube inserted into the substrateportion of the chip. Embodiments of sipper chips compatible with thepresent invention are described in U.S. Pat. No. 5,779,868, which isassigned to the assignee of the present invention. Commerciallyavailable sipper chips include chips used by the Caliper AMS 90 and 250HTS systems.

In most microfluidic devices, fluids are moved through the microchannelsof the device by means of electrokinetic forces, pressure forces, or acombination of the two. Electrokinetic forces are typically generated byapplying an electric field along the length of a microchannel, parallelto the desired direction of fluid flow. For example, in the microfluidicdevice 10 in FIG. 1, electrokinetic driving forces would be applied tothe microchannels, which are formed by enclosing the grooves 16, byplacing electrodes in the reservoirs 24 and applying voltages betweenthe various electrodes. Fundamental techniques for controllingelectrokinetic flow in the microchannels of a microfluidic device wereinvented by Dr. J. Michael Ramsey. These techniques are covered by aseries of issued and pending patents, including U.S. Pat. Nos. 6,001,299and 5,858,195. Dr. Ramsey's techniques control fluid flow withinmultiple microchannels can by simultaneously applying separatelycontrollable electric fields across the different microchannels.Software programs can be written for computer controlled power suppliesto generate highly specific and complex networks of flow within anetwork of microchannels.

This invention is directed to using ion-implantation to controllably andlocally increase the conductivity of a substrate or cover plate of amicrofluidic device. By increasing the conductivity in a defined area, apath for electrical current can be defined between microchannels. Thephysical separation distance between microchannels in a microfluidicdevice in accordance with the invention can range from between 10-100μm, and in most embodiments between 20 to 50 μm. The ion-implantationprocess provides control over the conductivity of the region of themicrofluidic device being implanted by controlling the dose, energy, andsubsequent thermal annealing of that region. Ion-implantation enableslocalized high-conductivity areas to be formed on a chip withoutdegrading performance elsewhere on the chip.

As will be described below, ion implanting according to the presentinvention can be performed in various areas or regions of the substrateand/or cover plates of a microfluidic device. When referencing the ionimplanting of an area or region, the terms “adjacent a microchannel” and“adjacent a reservoir”, or the like phrases, are used herein to mean avariety of possible relative positioning of the ion implanted area orregion and a microchannel and/or reservoir. As examples, “adjacent” canmean that the ion-implanted region: fully or partially overlaps (i.e.,is fully or partially integral) with a portion of a microchannel and/orreservoir; or is separated a distance from a portion of a microchanneland/or reservoir. Such a separation distance or overlap will beimplementation dependent.

Various authors have described methods to change the conductivity ofglass (or of fused silica) by ion implantation (see Okura, T., andYamashita, K, Solid State Ionics 136 SI:1049-1054 (2000); Rebohle, L.,et al., Applied Physics B-lasers and Optics 71:131-151 (2000); Nakajima,A., et al., J. Vacuum Sci. Technol. B 17:1317-1322 (1999); Hosono, H.,et al., J. Non-crystalline Solids 182:109-118 (1995); Prawer. S., etal., J. Appl. Physics 73:3841-3845 (1993); and Martin, P., et al., J.Appl. Physics 72:2907-2911 (1992)). Examples of implanted species thathave been used are: protons, sodium, antimony, silicon, germanium,carbon, titanium, and chromium. Ion implantation allows accurate controlof dose and depth, and has long been a vital part of silicon integratedcircuit technology, where such control is essential. The details of ionimplantation techniques, including thermal annealing and themanufacturing equipment to carry out ion implantation would be apparentto a person skilled in the relevant art.

To localize the effect of an ion-implantation process, some form ofmasking is typically used. This masking can be accomplished usinglithography techniques that employ either positive or negativephotoresist materials. Such lithography techniques are commonly used inthe integrated circuit industry, and have been applied in themanufacture of flat panel displays, circuit boards, microfluidicdevices, and various integrated circuits. When a lithography process isused to pattern a substrate, the substrate is first coated with one ormore layers of a photoresist material. In some embodiments, thesubstrate may be coated with a layer of chrome before the photoresist isapplied. The chrome may act as an adhesion layer between the photoresistand substrate materials to which the photoresist does not adequatelyadhere. The substrate is then placed in an aligner, in which thesubstrate is placed on a stage and held in place by a chuck. The chuckis typically a vacuum or electrostatic chuck capable of securely holdingthe substrate in place. The photoresist on the substrate is exposed toan image projected onto its surface by passing radiation through apatterned mask or reticle. As is known to those skilled in the relevantart, the radiation could be visible light, UV light, x-rays, ions, orelectrons.

The projected image produces changes in the characteristics of thecoating of photoresist material. These changes occur in the portions ofthe photoresist that were exposed to radiation during exposure.Subsequent to exposure, the layer is developed to produce a patternedlayer of photoresist. In some embodiments, the substrate covered withthe patterned layer of photoresist is then subjected to an etchingprocess. Some areas of the photoresist pattern expose the underlyingsubstrate from the etching process, while other portions of the patternshield the substrate from the etching process. Accordingly, thephotoresist pattern is effectively transferred to the underlyingsubstrate. As previously discussed, this combination of lithography andetching can be used to form grooves in a glass or polymeric substratethat, when covered, become the microchannels of a microfluidic device.In another aspect of the invention, a photoresist pattern formed by alithography process is used to shield portions of a substrate or coverplate from an ion implantation process.

As the size of a substrate increases, the equipment required to patternthe entire substrate at once becomes more expensive. So rather thanexpose the entire substrate at one time, sub portions of the photoresistlayer are exposed one at a time. A special type of aligner known as a“step-and-scan” aligner is designed to expose only a portion of asubstrate at a time. A step-and-scan aligner contains a projectionoptics system that has a narrow imaging slot. An entire substrate can beexposed by placing the imaging slot and reticle over different portionsof the substrate. To accomplish this, the stage on which the wafer sitsis then moved between exposures to allow multiple copies of the reticlepattern to be exposed over the substrate surface. In this manner, thesharpness of the image projected onto the substrate is maximized. Usinga step-and-scan technique generally assists in improving overall imagesharpness. For more background see, Nonogaki et al., “MicrolithographyFundamentals in Semiconductor Devices and Fabrication Technology”(Marcel Dekker, Inc.: New York, N.Y. 1998). Step-and-repeat and fieldstitching lithography techniques can also be used.

An exemplary method for modifying the conductivity of a portion of amicrofluidic device is shown in FIG. 2. This method employs thelithography and ion implantation processes described above. For clarity,the method in FIG. 2 will be described in terms of its application tothe glass substrate portion of a microfluidic device. One skilled in therelevant art would recognize that methods in accordance with theinvention could be applied to other portions of a microfluidic device,such as a cover plate, and could be applied to substrates and coverplates made of materials other than glass.

The first step in the method of FIG. 2 is the fabrication of the patternof grooves in the substrate that will, when covered, form themicrofluidic channels in a microfluidic device. The pattern of groovesis formed in step 202 by means of the combination of lithography andetching described above. The substrate is made of an insulating materialsuch as glass, a silica-based material or a polymeric-based material.Before proceeding to the next step, any residual layers, such as chromeand photoresist, are removed using known techniques.

In a step 204, a thick photoresist such as Shipley SPR 220 or ClariantAZ EXPLOF 5000 is applied and patterned by exposing and developing thephotoresist. In some embodiments it may be advantageous to soft bake thephotoresist after it is applied to the substrate. The portions of thislayer of photoresist that are not removed during the developing processwill shield the portions of the substrate they cover from the ionimplantation process in a step 206. In some embodiments it may beadvantageous to hard bake the photoresist after it is developed. Duringion implantation 206, the exposed portions of the substrate, i.e. theportions of the substrate not covered by photoresist, will have theirconductivity increased by the implantation of ions. The portions of thesubstrate with increased conductivity regions are also referred toherein as “glass resistors.” The degree to which the conductivity of theglass resistors increases during ion implantation depends on the doseand energy of the implanted ions. After ion implantation is complete,the photoresist layer is stripped away 208. Due to possible hardening ofthe photoresist during ion implantation, especially during after highdose implants, stripping the photoresist 208 may require plasmatreatments in addition to the standard wet chemical baths. As would alsobe apparent to a person skilled in the relevant art, it may be desirableto thermally anneal the substrate to repair damage to the substrate andto electrically activate the implanted ions. Finally, at a step 210 thesubstrate, which now comprises a pattern of grooves and one or moreglass resistors, is bonded to a cover plate.

To completely avoid the problems associated with hardening ofphotoresist during ion implantation, a material other than photoresistmay be used to shield portions of the substrate from the ionimplantation process. In other words, a layer of material could bedeposited on the substrate to form a masking layer. The material formingthe masking layer could be a metal, or an insulator material differentthan the substrate material. Specific examples of materials suitable foruse as a masking layer are chrome, silicon nitride, amorphous siliconand polysilicon. The masking layer would form a pattern that covers theportions of the substrate that are to be shielded from theion-implantation process, and leaves exposed the portions of thesubstrate that are to be implanted with ions. The masking layer istypically patterned by means of a lithography process. In an exemplaryembodiment, a layer of photoresist is patterned (i.e. applied,developed, and exposed) so that only the portions of the substrate thatare to be covered by the masking layer are left exposed. Next, a layerof chrome is sputtered onto the substrate. Finally, the resist isstripped so that only the chrome deposited on the exposed areas of thesubstrate remains. The process used to deposit chrome in this exemplaryembodiment is commonly known as a lift-off process. After the ionimplant is carried out, the masking layer can be removed using aselective etch process so as not to affect the underlying substrate.

A second exemplary method for modifying the conductivity of a portion ofa microfluidic device is shown in FIG. 3. In the embodiment of FIG. 3the substrate is subjected to ion-implantation before the pattern ofgrooves is formed on the substrate surface. In a step 302 shallowalignment marks are etched at the edges of a substrate. These alignmentmarks facilitate the proper alignment of the ion-implanted regions, theglass resistors, with the yet-to-be formed pattern of grooves. In a step304, a layer of photoresist is applied to the substrate, and is thenexposed and developed. This layer of photoresist, just like the layer ofphotoresist in step 204 of FIG. 2, serves to shield portions of thesubstrate from the ion implantation process. As was discussed withregards to the layer of photoresist in FIG. 2, the photoresist in step304 of FIG. 3 may harden during the ion implantation process.Accordingly, in some embodiments it may be desirable to replace thelayer of photoresist in step 304 with a masking layer. In a step 306,ion implantation is performed to form one or more glass resistors. In astep 308 the photoresist is stripped. As discussed with regards to theembodiment in FIG. 2, it may be desirable to thermally anneal thesubstrate after implantation. In a step 310 a second layer photoresistis applied to substrate, and is then exposed and developed. This layerof photoresist defines the pattern of grooves that will form themicrochannels in the finished microfluidic device. In a step 312, thepattern of grooves is etched into the surface of the substrate. In afinal step 314, the photoresist is stripped from the substrate. Thesubstrate and cover plate can then be bonded together to form amicrofluidic device.

In other embodiments of the invention, the conductivity of portions ofthe cover plate are modified by subjecting the cover plate to an ionimplantation process such as one of those shown in FIGS. 2 and 3. Instill other embodiments, the conductivity of portions of both the coverplate and the substrate are modified.

Glass resistors formed by ion implantation according to the presentinvention can be used is a variety of ways in microfluidic devices. Oneexemplary benefit that can be achieved by employing glass resistors in amicrofluidic device is increased sensitivity in a protein assay. Glassresistors in accordance with the present invention can employed in aprotein assay chip in the Agilent 2100 Bioanalyzer, for example.

FIG. 4 illustrates a 16-well protein assay microfluidic chip 400 inaccordance with the present invention. The wells or reservoirs 406, 408,416-436, 440, 450, 452, 484 are in fluid communication with the networkof microchannels, which includes microchannels 460-470. Of the sixteenwells, ten 416-436 contain samples to be analyzed, two 408, 484 arewaste wells, one 440 supplies a reagent such as a fluorescent dye thatenables detection of selected species, and one 406 is a source ofbuffer. These fourteen wells are fluidly connected by a first network ofchannels. The remaining two wells 450,452 are fluidly connected by asecond network of channels. In this embodiment, the first and secondnetworks of channels are not fluidly connected. A region of increasedconductivity, a glass resistor, is shown generally at a region 490. FIG.5 is a magnified view of the region adjacent the glass resistor 490,which is enclosed by a box for illustrative purposes.

The basic function of chip 400 is to separate a sample into itscomponents by electrophoretic means. Means of electrophoretic separationthat may be employed in embodiments of the invention are described inU.S. Pat. No. 5,948,227, which is assigned to the assignee of thepresent invention. In the device of FIGS. 4 and 5, the electrophoreticseparation takes place in separation channel 404. Samples from wells416-424 are injected into separation channel 404 via channels 462 and472, while samples from wells 428-436 are injected into separationchannel 404 via channels 464 and 472. The electrophoretic separation ofthe sample takes place as the sample travels through the separationchannel 404 from the intersection of channels 472 and 404 to waste well408. In this exemplary embodiment, only one sample at a time isseparated in the separation channel 404. To improve device throughput,however, a sample can be preloaded into channel 472 while a previouslyinjected sample is being separated in separation channel 404. Thepreloading process does not interfere with the separation taking placein separation channel 404 because the flow of the sample being preloadedis diverted into channel 460, which empties into waste well 484.Preloading a subsequent sample in this manner minimizes the timerequired to load the sample into the separation channel 404. This typeof preloading is described in more detail in U.S. Pat. No. 5,948,227,and is implemented in DNA and RNA assay chips for the Agilent 2100Bioanalyzer.

To inject a sample into the separation channel 404, the sample travelsfrom its well through either channel 462 or 464 into channel 472. Thesample is propelled through those channels by electrokinetic forcesgenerated by voltages applied between electrodes (not shown) immersed inthe sixteen reservoirs of the microfluidic device 400. When the samplearrives at the “T” intersection between channels 472 and 404, the sampleis directed to travel straight out of channel 472, across channel 404,into the opposite wall of channel 404. The electrical current directingthe flow of sample into the wall flows through channel 472, through theglass resistor 490, and finally through channel 468. The glass resistorallows electrical current to flow from channel 472 into channel 468,even though channels 472 and 468 are not in fluid communication. Thedesired electrical currents are supplied via power-supply electrodesimmersed in the sixteen reservoirs of the microfluidic device 400. If,for example, the sample being injected into separation channel 404originated from reservoir 418, the desired electric current would begenerated by applying appropriate voltages to electrodes in reservoirs418, 450, and 452. Note that voltages may have to be simultaneouslyapplied to other reservoirs connected to the first channel network toprevent the flow of other samples into channels 462, 464 and 472, and toprevent the diversion of sample into waste well 484. Also, a voltage mayhave to be applied across the length of separation channel 404, by meansof voltages applied to electrodes in reservoirs 406 and 408, to preventnet fluid movement along the separation channel during injection. Powersupplies capable of supplying the voltages and currents required toimplement this and other embodiments of the invention are described inU.S. Pat. No. 5,965,001, which is assigned to the assignee of thepresent invention.

As the sample is injected from channel 472 toward the opposing wall ofthe “T” intersection between channels 472 and 404, the sampleaccumulates in the portion of channel 404 near the intersection,providing a more concentrated sample. The longer the sample isconcentrated in this manner, the more sample will be available forseparation and detection. The degree of concentration appropriate for agiven analysis will represent a simple trade-off between loading timeand sensitivity. A “high sensitivity” script can be run on the same chipas a “normal” script, the only difference being a longer loading timeand therefore total analysis time. By way of further example and notlimitation, the techniques described herein can be used in connectionwith the inventions disclosed in Caliper U.S. Pat. Nos. 5,976,336 and6,153,073, both of which are assigned to the assignee of the presentinvention.

The increased conductivity of region 490 allows an electrical potentialto be maintained across the blind “T” intersection, enabling the flow offluid from channel 472 to be directed into a wall. The increase inconductivity can be set based on the separation between the channelsbetween which the glass resistor provides a conductive path, and thelevel of ion implantation in the glass resistor. The amount ionimplantation can be determined by the dose amount, the energy level usedduring implantation, and the thermal annealing process. In an exemplaryembodiment, the sheet conductivity of a glass substrate, as measured bya four point probe, is about 10¹⁵ ohms per square (Ω/

). Ion implantation can increase the glass conductivity by severalorders of magnitude, for example up to about 10⁸ Ω/

. Prawer et al., supra, shows that a sheet resistance of 10⁸ Ω/

can be achieved by implanting carbon at a dose of 10¹⁶ ions/cm². This isa high dose, but is attainable using standard implantation methods knownin the art. This level of sheet resistance is suitable for the presentinvention, as can be seen by the following example.

A microfluidic device similar to that shown in FIGS. 4 and 5 with 40 μmwide channels and with a gap of 100 μm between channel 490 and channels466 and 468 can be manufactured using the methods described above. Inthis exemplary embodiment, the glass resistor electrically connectingchannel 490 and channel 466 and 468 could have a length to width ratioof about 2 (allowing for some lateral current spreading). Assuming asheet resistance of 10⁸ Ω/

, the resistance of the glass resistor would be 2×10⁸ ohms. If thecurrent used to inject the sample were about 3 μA, which is typical ofmicrofluidic devices in accordance with the invention, the voltage dropacross the glass resistor would be 600V. This is an easily achievablevalue, since the high voltage power supplies used in electrophoreticmicrofluidic analysis systems typically supply voltages in the range of1500V to 3000V, as is the case with the Agilent 2100 Bioanalyzer, andthe Caliper Technologies AMS-90 systems.

Exemplary Methods

Various methods can use microfluidic devices in accordance with thepresent invention. Such methods include, but are not limited toseparating macromolecules by capillary electrophoresis and detectingreactions. Such methods employ a microfluidic device comprising aninsulating substrate, at least one of a microchannel and a well formedin the insulating substrate, and at least one ion implanted region inthe insulating substrate located at or adjacent the at least one of themicrochannel and the well, the at least one ion implanted region havingincreased conductivity compared to the insulating substrate.

A method of separating macromolecules by capillary electrophoresisaccording an embodiment of the present invention comprises: providing amicrofluidic device, as described above; introducing a sample containingthe macromolecules into one end of the microchannel; and applying avoltage gradient across the length of the microchannel, whereby themacromolecules in the sample are separated in the microchannel.

A method of detecting a reaction according an embodiment of the presentinvention, comprises the steps of: introducing a first reagent into amicrochannel of the microfluidic device; introducing a second reagentinto the microchannel, whereby the first and second reagents mixtogether to form a reagent mixture; introducing a test compound into thereagent mixture; and detecting an effect of the compound on the reagentmixture.

CONCLUSION

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample, and not limitation. It will be apparent to persons skilled inthe relevant art that various changes in form and detail can be madetherein without departing from the spirit and scope of the invention.This is especially true in light of technology and terms within therelevant art(s) that may be later developed. For example, the presentinvention is applicable in a variety of applications, such as electricalconnectors for application of electrical energy, as in-line sensors forsensing conductivity, temperature, or the like. Alternatively, increasedconductivity regions can be used on chips to apply or modify electricfields for various purposes. By way of further example and notlimitation, the techniques described herein can be used in connectionwith the inventions disclosed in Caliper U.S. Pat. No. 5,965,410, whichis assigned to the assignee of the present invention.

The present invention has been described above with the aid offunctional building blocks, modules or steps illustrating theperformance of specified functions and relationships thereof. Thecollection of sub-steps or boundaries of these functional buildingblocks have been defined herein for the convenience of the description.Alternate boundaries can be defined so long as the specified functionsand relationships thereof are appropriately performed. Any suchalternate collection of sub-steps or boundaries are thus within thescope and spirit of the claimed invention. Thus, the breadth and scopeof the present invention should not be limited by any of theabove-described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents. Allpublications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

1. A microfluidic device, comprising: a first layer made of aninsulating material; a first microchannel formed in said first layer; atleast one of a second microchannel and a well formed in said firstlayer; and at least one ion implanted region in said first layer locatedat or adjacent the first microchannel and said at least one of saidsecond microchannel and said well, said at least one ion implantedregion providing a conductive path between said first microchannel andsaid at least one of the second microchannel and the well.
 2. The deviceof claim 1, wherein said insulating material comprises a silica-basedmaterial.
 3. The device of claim 1, wherein said insulating materialcomprises a polymer material.
 4. The device of claim 1, wherein saidfirst layer comprises a substrate of the microfluidic device.
 5. Thedevice of claim 1, further comprising a second layer bonded to saidfirst layer.
 6. The device of claim 1, wherein said first layercomprises a cover plate.
 7. The device of claim 1, wherein saidinsulating material comprises a silica-based material.